Cloning of DNA fragments

2.2.5.1 Polymerase chain reaction (PCR)

DNA fragments were amplified from various plasmid and genomic sources. A standard PCR reaction in a total volume of 50 μl was prepared as followed:

30-35 μl H2O (sterile double distilled)

5.0 μl 10X PCR-buffer

1.5 μl 50 mM MgCl2

2.0 μl Forward-primer (10 pmol/μl) 2.0 μl Reverse-primer (10 pmol/ μl)

1.0 μl 10 mM dNTPs

1.0 μl plasmid DNA (5 ng/μl) or genomic DNA (50-100 ng/ μl) or a bacterial colony

0.5 μl Taq-polymerase

Reactions were homogenized and the PCR was performed in a TRIO-thermoblock (Biometra, Göttingen, Germany). The optimal number of PCR cycles and the annealing temperature was determined empirically for each PCR. A standard PCR programme was as followed:

94°C 5 min of denaturing

94°C 30 sec (30 times) of denaturing TA 30 sec (30 times) of primer binding 72°C 45 sec (30 times) of elongation 72°C 5 min for final extension

4°C for keeping the samples stable until they are collected.

TA = annealing temperature = TM ± 4 °C

TM = melting temperature of the primers. For primers with different TM, the lower one is considered for the calculation of the TA.

2.2.5.2 Restriction endonuclease treatments

DNA digestion was carried out by restriction endonucleases according to the following criteria: the reaction buffer (10X) was 1/10 of the end volume and 5 U of restriction enzymes was used per 1 μg of DNA to digest. A double digestion was possible per reaction only when

both restriction enzymes can be active in the same buffer; otherwise the digestions were performed sequentially.

2.2.5.3 Dephosphorylation

Linear plasmid vectors were dephosphorylated at their 5’ end with shrimp alkaline phosphatase (SAP; Boehringer/Roche, Mannheim, Germany) in order to avoid self-ligation of their cohesive or compatible ends. The reaction was made in 10 μl reaction comprising 1 μl of 10X SAP buffer, 1.0 μl (1 unit) SAP, and adequate amount of the plasmid vector. The mixture was brought to 10 μl with sterile distilled water. The reaction was incubated for 10 min at 37°C followed by inactivation of SAP at 65°C for 15 min.

2.2.5.4 Ligation

To generate recombinant plasmid DNA constructs, the insert-DNA was ligated to a linear vector in a ligation reaction. The ligation was performed in 20 μl reaction volume containing 1X ligase buffer, x μl digested and purified plasmid DNA vector, 1.0 μl T4 DNA ligase (MBI-Fermentas; St. Leon-Rot, Germany or Invitrogen/GibcoBRL; Karlsruhe, Germany), and y μl insert-DNA. The mixture was brought to 20 μl with sterile H2O and incubated at 16°C for 20 h or according to the ligase manufacturer. For an optimal ligation reaction the molar amount of plasmid vector should be the third of the insert-DNA in the reaction.

2.2.5.5 Transformation

2.2.5.5.1 Calcium-competent E. coli

A bacterial culture (100 ml) was allowed to grow (37°C, 250 rpm) till OD600 = 0.5, cooled down on ice for 5 min and centrifuged (5 min, 5000 rpm, 4 °C). The pellet was suspended in 1 ml pre-chilled 0.1 M CaCl2 and centrifuged once again as above. The pellet was resuspended in 9.0 ml pre-chilled 0.1 M CaCl2 and centrifuged as above. The pellet was finally resuspended in 1 ml pre-chilled 0.1 M CaCl2 + 15% (v/v) glycerol and stored at -70°C in aliquots of 100 μl of competent cells.

2.2.5.5.2 Transformation of calcium-competent E. coli

One µl plasmid DNA (5-10 ng/μl) or 1-5 µl of the ligation product was added to one aliquot of calcium-competent cells (100 μl) and carefully mixed. The mixture was incubated on ice for 1 h and heat-shocked in a water bath at 42 °C for 45 seconds. Cells were diluted with 650 μl LB medium and incubated under agitation (250 rpm) at 37°C for 1 h. Aliquots

(100-200 μl) of the cell suspension were then spread on selective agar-plates and incubated at 37ºC overnight.

2.2.5.5.3 Preparation of electrocompetent E. coli

E. coli bacteria were inoculated in 10 ml LB medium and pre-cultured overnight (37°C, 250 rpm). On the following day, the pre-culture was used to start a fresh culture in 200 ml LB medium till OD600 = 0.6. The culture was then cooled down on ice for 30 min and centrifuged (5 min, 5000 rpm, 4°C). The cell pellet was washed in 50 ml pre-chilled sterile H2O, centrifuged as above, re-washed in 25 ml of H2O and centrifuged again. The pellet was further washed twice in 25 ml 10% (v/v) glycerol and resuspended in 10 ml cold GYT-medium. After centrifugation, the cell pellet was resuspended in 2 ml GYT. Aliquots (50 μl) of competent cells were shock-frozen in liquid nitrogen and stored at -70°C.

GYT: 10% (v/v) glycerol, 0.125% (w/v) selected yeast extract, 0.25% (w/v) trypton.

2.2.5.5.4 Preparation of electrocompetent A. tumefaciens

A. tumefaciens was inoculated in 3 ml YEB-Rif medium and pre-cultured for overnight (28°C, 250 rpm). Cells were pelleted, diluted into fresh YEB-Rif (50 ml) and further cultured till OD600 = 0.5. The cell culture was cooled down on ice for 30 min and centrifuged (5000 rpm, 4 °C) for 5 min. The pellet was resuspended in cold sterile H2O and centrifuged as above. The cells were further resuspended in the following solutions with centrifugations (5000 rpm, 10 min, 4 °C) between the suspensions, as follows:

25 ml 1 mM Hepes pH 7.5

12.5 ml 1 mM Hepes pH 7.5

10 ml 10% (v/v) glycerol, 1 mM Hepes pH 7.5 5 ml 10% (v/v) glycerol, 1 mM Hepes pH 7.5

2 ml 10% (v/v) glycerol

1 ml 10% (v/v) glycerol

Aliquots (40 μl) of the last suspension of electrocompetent cells were made, shock-frozen in liquid nitrogen and stored at -70°C.

2.2.5.5.5 Transformation via electroporation (Tung and Chow 1995)

Aliquots of electro-competent cells were thawed on ice before transformation. 1 μl DNA of the ligation product or plasmid DNA (approximately 5-10 ng/μl) was added to the electro-competent cells and carefully mixed in a pre-chilled Electro–cuvette (Bio-Rad, Germany).

The DNA was brought into the cells by electroporation after a single pulse of 3 to 5 sec (GenePulser II, Bio-Rad). Cells were immediately diluted in 1 ml YEB-mediun or 800 μl SOC medium and cultured for another 1 h at 37ºC (for E. coli) or 2-3h at 28ºC (for A.

tumefaciens) under agitation at 250 rpm. 100 μl aliquots of the cell culture were spread on selective media and incubated at appropriate temperatures (Table 3).

Table 3 Electroporation parameters of E. coli and A. tumefaciens cells

Transformation parameters E. coli A. tumefaciens

DNA Ligated vector or plasmid Only plasmid

Electro–cuvette 1 mm 2 mm

Capacity 25 μF 25 μF

Power 1.6 kV 2.5 kV

Resistance 200 Ω 400 Ω

Incubation Medium SOC YEB

Incubation temperature 37ºC 28ºC

Incubation time for selecting clones 12-16 h 48-72 h

2.2.5.5.6 Biolistic transformation of Arabidopsis leaves

The preparation of the microcarriers and DNA coating were performed according to Sanford et al. (1993) with a few modifications. Gold particles (1.6 µm diameter) were used as microcarriers for the bombardment and prepared as follows: 30 mg gold particles were weighted into a 1.5 ml microfuge tube and vigorously vortexed in 1 ml of 70% ethanol (v/v) on a platform vortexer for 3-5 min. The particles were further allowed to soak in the ethanol solution for 15 min and spun down (10,000 rpm) for 10 seconds in a microfuge. The supernatant was discarded and the gold particles were washed three times as follows: vigorous vortexing for 1 min in 1 ml sterile water, 1 min pause to let the particles to settle, recovery of particles by briefly spinning in a microfuge and removal of the liquid. After the third wash, 500 µl sterile 50% (v/v) glycerol were added to the particles to bring their concentration to 60 mg/ml, assuming no loss during the preparation. Prepared gold particles were stored at 4°C in 50 µl aliquots for up to one month without decrease in the transformation efficiency. Fifty microlitres of microcarriers were used for the coating procedure: 15 µl plasmid-DNA

(1 µg/µl), 50 µl of 2.5 M CaCl2 and 20 µl of 100 mM freshly prepared spermidine (Sigma S0266, Munich, Germany) were in this order added to the gold suspension while vortexing for 3 min at maximum speed. The suspension was briefly centrifuged as above and the liquid discarded. The particles were washed with 140 µl 70% (v/v) ethanol, spun down, re-washed with 140 µl 100% ethanol and mixed, after centrifugation, with 30 µl 100% ethanol. The particles were gently resuspended by tapping the side of the tube several times then by vortexing at low speed for 2-3 seconds. Fifteen microlitres of the gold suspension were used for each bombardment.

The bombardment procedure followed the PDS-1000/He manufacturer’s instruction (Bio-Rad, Munich, Germany). Briefly, a plastic macro-carrier disk containing 15 μl of DNA-coated gold particle (micro-carrier) suspension was placed into the macro-carrier holder along with a stopping metal grid. The system macrocarrier-microcarrier-stopping grid was placed into the launch assembly unit, as described by the manufacturer. Well-expanded Arabidopsis leaves were arranged on a plate of solid MS medium and placed at 5-10 cm below the stopping screen. Vacuum was then applied to increase the gas pressure within the bombardment chamber. The release of the pressure led to the burst of the rupture disk and allowed the macro-carrier to eject at high celerity the DNA-coated gold particles into the leaves. The particles were accelerated with a helium pressure of 1150 pounds per square inch (psi) under a vacuum of 27 mm Hg (3.6 MPa). After bombardment the leaves were incubated in water overnight and analysed under a confocal laser microscope within 48 h.

2.2.5.5.7 A. tumefaciens-mediated transient transformation of Arabidopsis seedlings:

FAST assay (Li et al. 2009)

Ten to twelve day-old Arabidopsis seedlings were transiently transformed by the FAST (Fast Agrobacterium-mediated Seedling Transformation) technique based on the co-cultivation of Agrobacterium cells (GV3101::pMP90) harbouring the transgene in a binary vector with the seedlings in a medium containing the surfactantSilwet L-77. One day before co-cultivation, a single colony of A. tumefaciens was inoculated into 2 mL of YEB medium with appropriate antibiotics(50 µg/ml kanamycin and 50 µg/ml rifampicin) and cultured at 28°C for 18-24 h.

On theday of co-cultivation, the saturated Agrobacteriumculture was diluted toOD600= 0.3 in 10 mL of fresh YEB medium without antibiotics. The cells were further grown at 28°C under vigorous agitation until the OD600 readingreaches 1.5-2. After centrifugation at 6000 g for 6 min, the cell pellet was resuspended in 10 mL of washing solution (10 mM MgCl2).The cell suspension was again pelleted by centrifugationat6000 g for 5 min and resuspended in 1 mL

washing solution as above. 30 to 50 seedlings were carefully transferred from plates into a clean 100 x 20 mmPetri dish filled with 20 mL of co-cultivationmedium (1/4 MS, 0.005%

Silwet L-77). Agrobacterium cell suspension was added to the co-cultivation mediumto a final density of OD600 = 0.5and mixed well by gentle shaking. The Petridish was wrapped with aluminum foil and incubated in theplant growth chamberfor 36-40 h. Plates were kept without aluminum foil in the case of ALDH7B4 promoter study, as this gene is induced under prolonged dark conditions. After the co-cultivation period,the medium wasreplaced with the surface sterilizationsolution (0.05% sodium hypochlorite) and incubated for 10 min,washed three times with H2O to remove epiphyticbacteria. Seedlings were finally incubated in 0.5X MS, 500 µg/mL carbenicillin to inactivateremaining Agrobacteriumcells prior applying the stressors.

2.2.5.5.8 A. tumefaciens-mediated stable transformation of Arabidopsis plants

Arabidopsis transgenic plants were generated via A. tumefaciens-mediated transformation of wild-type (Col-0) plants according to Clough and Bent (1998). The Agrobacterium clone carrying the transgene was cultured (28ºC, 250 rpm) in 250 ml YEB/kanamycin/rifampicin (50 µg/mL kanamycin and 50 µg/mL rifampicin) until OD600 = 0.7 – 0.8. The cell suspension was added with 0.05% (v/v) of the surfactant Silwet L-77 and collected in a 500 ml beaker.

Flowering plants with young inflorescences and un-opened flowers were carefully inverted and immersed in the infiltration medium with gentle rotation for 20 sec. Care was taken to immerge all the inflorescences in the solution. Dipped plants were thereafter returned to trays and covered with plastic bags. A few holes were made on the bags for ventilation. Three days after infiltration the plastic bags were removed and the plants were supported with wooden sticks and grown until they set the first generation of seeds (T1).